Expression analysis of protein inhibitor of activated STAT (PIAS) genes in IFNβ-treated multiple sclerosis patients

Figures & data

Table 1 Nucleotide sequence of primers and probes

Gene name	Primer and probe sequence	Primer and probe length	Product length
HPRT1	F: AGCCTAAGATGAGAGTTC	18	88
	R: CACAGAACTAGAACATTGATA	21
	FAM -CATCTGGAGTCCTATTGACATCGC- TAMRA	24
PIAS1	F: AGCCTAAGGGAAAGCCATAGC	21	83
	R: ATGTCTGGTATGATGCCAAAGATG	24
	FAM-TGCCGTGTCCGTGCTGCTCCTGT- TAMRA	24
PIAS2	F: CACGAACTCTTGAAGGACTTTCTG	24	126
	R: AAGTGGAAGGCAACGAGTGG	20
	FAM - CCAGCCACGGCCAAGTCAGGTTCT -TAMRA	24
PIAS3	F:GCCCTACCTGGAAGCAAAGG	20	120
	R: GTACTCATGTAGTGGGAGACTGG	23
	FAM- CCCACCCAACGTGCCCATAGCAGG - TAMRA	24
PIAS4	F: GAGAAGAAGCCCACCTGGATG	21	77
	R: AGGAGCCCGTCGATGATGAG	20
	FAM- CCCGTGTGCGACAAGCCAGCCC - TAMRA	22

Table 2 Characteristics of study participants

Variables	MS patients (n=48)	Healthy subjects (n=52)
Female/male (no., %)	39 (81.2%)/9 (18.7%)	37 (71.1%)/15 (28.8%)
Age (mean ± SD, Y)	36.2±2.9	35.3±2.1
Age range (Y)	17–55	22–60
Age of onset (mean ± SD, Y)	31.41±2.8	–
Relapsing–remitting course (no., %)	100 (100%)	–
Disease duration (mean ± SD, Y)	4.58±3.2	–
EDSS (mean ± SD)	3.07±2.7	–

Abbreviations: EDSS, Expanded Disability Status Scale; MS, multiple sclerosis; Y, years.

Table 3 PIAS1 expression in MS patients compared with healthy subjects

		Control numbers	MS patient numbers	Expression ratio	ΔΔCT	SD	P-value	95% highest density interval
Total		52	48	1.1566	0.205	−0.23	0.163	−0.23 to 0.63
Male		15	9	1.2754	0.495	−0.58	0.29	−0.6 to 1.5
Female		37	39	1.1270	0.123	−0.12	0.304	−0.364 to 0.62
<30	Male	2	0	–	–	–	–	–
	Female	8	9	0.8340	−0.352	0.99	0.744	−2.24 to 1.61
30–40	Male	2	3	0.9361	–	–	–	–
	Female	7	15	1.4601	0.549	−0.06	0.253	−0.66 to 1.7
>40	Male	11	6	1.3332	0.53	−0.83	0.325	−1.25 to 2.28
	Female	22	15	1.1821	0.327	−0.34	0.25	−0.339 to 1

Abbreviation: MS, multiple sclerosis.

Table 4 PIAS2 expression in MS patients compared with healthy subjects

		Control numbers	MS patient numbers	Expression ratio	ΔΔCT	SD	P-value	95% highest density interval
Total		52	48	0.7710	−0.552	−0.28	0.06	−1.06 to −0.02
Male		15	9	0.6585	−0.822	−0.32	0.093	−2 to 0.3
Female		37	39	0.8108	−0.459	−0.3	0.234	−1.07 to 0.15
<30	Male	2	0	–	–	–	–	–
	Female	8	9	0.5381	−1.17	−0.66	0.19	−3.23 to 0.894
30–40	Male	2	3	0.7113	–	–	–	–
	Female	7	15	1.1310	0.055	0.46	0.733	−1.5 to 1.6
>40	Male	11	6	0.5846	−1.7	−1.21	0.116	−3.4 to 1.23
	Female	22	15	0.8144	−0.493	−0.449	0.336	−1.4 to 0.43

Abbreviation: MS, multiple sclerosis.

Table 5 PIAS3 expression in MS patients compared with healthy subjects

		Control numbers	MS patient numbers	Expression ratio	ΔΔCT	SD	P-value	95% highest density interval
Total		52	48	0.8615	−0.397	−0.43	0.485	−1.05 to 0.26
Male		15	9	1.1217	0.217	−0.86	0.646	−0.95 to 1.47
Female		37	39	0.7977	−0.53	−0.23	0.403	−1.34 to 0.28
<30	Male	2	0	–	–	–	–	–
	Female	8	9	0.6876	−0.83	−1.16	0.583	−3.54 to 1.93
30–40	Male	2	3	1.1799	–	–	–	–
	Female	7	15	1.3370	0.393	0.44	0.422	−1.3 to 2.1
>40	Male	11	6	1.1479	0.258	1.6	0.683	−2.07 to 2.56
	Female	22	15	0.6357	−0.825	−0.25	0.253	−2.08 to 0.42

Abbreviation: MS, multiple sclerosis.

Table 6 PIAS4 expression in MS patients compared with healthy subjects

PIAS 4		Control numbers	MS patient numbers	Expression ratio	ΔΔCT	SD	P-value	95% highest density interval
Total		52	48	1.1912	0.371	−0.22	0.226	−0.302 to 1.08
Male		15	9	1.3012	0.644	−0.65	0.45	−0.99 to 2.31
Female		37	39	1.1740	0.324	−0.08	0.301	−0.46 to 1.08
<30	Male	2	0	–	–	–	–	–
	Female	8	9	0.7655	−0.614	−0.34	0.716	−3.15 to 2.04
30–40	Male	2	3	1.1178	–	–	–	–
	Female	7	15	1.1824	0.206	1.33	0.707	−2.2 to 2.6
>40	Male	11	6	1.4277	0.797	−0.96	0.394	−1.7 to 3.28
	Female	22	15	1.3703	0.6	−0.41	0.174	−0.46 to 1.7

Abbreviation: MS, multiple sclerosis.

Figure 1 Relative expression levels of PIAS genes in multiple sclerosis patients compared with healthy subjects.

Note: Each sex-/age-based subgroup of patients are compared with the corresponding subgroup of healthy subjects.

Table 7 The results of spearman correlation analysis between PIAS genes expressions and clinical data

	PIAS1				PIAS2				PIAS3				PIAS4
	Age	EDSS	Age at onset	Disease duration	Age	EDSS	Age at onset	Disease duration	Age	EDSS	Age at onset	Disease duration	Age	EDSS	Age at onset	Disease duration
Male	−0.8	−0.66*	−0.14	0.09	−0.29	−0.55	0.07	−0.1	−0.14	−0.77**	−0.08	0.1	0.01	−0.77**	−0.35	0.29
Female	−0.16	0.24	−0.33*	0.1	−0.04	−0.29	−0.21	0.26	−0.07	−0.14	−0.22	0.3	0.02	−0.19	−0.29	0.28
Case	−0.25	−0.35*	−0.28*	0.16	−0.02	−0.3*	−0.14	0.23	−0.08	−0.3*	−0.24	0.3*	−0.18	−0.34*	−0.31*	0.35*
Control	−0.12	−	−	−	−0.05	−	−		0.76**	−	−	−	−0.00	−	−	−

Notes:

** Correlation is significant at the 0.01 level;

* correlation is significant at the 0.05 level.

Abbreviation: EDSS, Expanded Disability Status Scale.

Table 8 Comparison of PIAS genes expression levels between male and female subjects

			PIAS1				PIAS2				PIAS3				PIAS4
Study groups	Male numbers	Female numbers	Expression ratio	SD	P-value	95% highest density interval	Expression ratio	SD	P-value	95% highest density interval	Expression ratio	SD	P-value	95% highest density interval	Expression ratio	SD	P-value	95% highest density interval
Total	50	50	0.09	0.29	0.559	−0.49 to 0.64	−0.05	0.34	0.147	−0.7 to 0.62	0.17	0.47	0.482	−0.74 to 1.09	0.57	0.43	0.136	−0.29 to 1.42
Case	11	39	0.18	0.46	0.348	−0.72 to 1.06	−0.31	0.56	0.735	−1.39 to 0.85	0.63	0.7	0.491	−0.71 to 2.03	0.56	0.66	0.162	−0.75 to 1.87
Control	13	37	−0.04	0.37	0.495	−0.76 to 0.69	0.21	0.4	0.366	−0.55 to 0.99	−0.31	0.67	0.169	−1.57 to 1.02	0.54	0.56	0.294	−0.58 to 1.62
<50	12	59	0.37	0.41	0.472	−0.45 to 1.15	−0.12	0.48	0.079	−1.06 to 0.81	0.5	0.56	0.146	−0.6 to 1.61	0.55	0.72	0.51	−1.03 to 1.79
>50	12	17	−0.11	0.34	0.137	−0.78 to 0.53	−0.1	0.47	0.084	−1 to 0.85	−0.4	0.89	0.779	−2.23 to 1.33	0.32	0.64	0.476	−0.95 to 1.55

Notes: Reference group: male, P-values computed from nonparametric frequentist methods.

Table 9 Results of median regression for analysis of association between PIAS genes expression and independent variables in patients

Variables	PIAS1					PIAS2					PIAS3					PIAS4
	Beta	SE	t	P-value	95% CI	Beta	SE	t	P-value	95% CI	Beta	SE	t	P-value	95% CI	Beta	SE	t	P-value	95% CI
Gender (M/F) EDSS Age Disease duration Age at onset	−0.64	0.69	−0.93	0.356	−2.02 to 0.75	−0.05	0.69	−0.07	0.942	−1.44 to 1.34	−0.64	0.92	−0.7	0.491	−2.48 to 1.21	−0.41	1.07	−0.38	0.706	−2.57 to 1.76
	−0.12	0.19	−0.63	0.529	−0.5 to 0.26	−0.2	0.19	−1.07	0.289	−0.58 to 0.18	−0.33	0.25	−1.33	0.192	−0.83 to 0.18	−0.56	0.3	−1.89	0.065	−1.14 to 0.04
	−0.04	0.05	−0.78	0.44	−0.13 to 0.06	−0.01	0.05	−0.15	0.879	−0.1 to 0.09	−0.13	0.07	−2.07	0.045	−0.25 to−0.01	−0.06	0.08	−0.83	0.412	−0.21 to 0.09
	−0.04	0.05	−0.76	0.451	−0.14 to 0.07	0.03	0.05	0.55	0.588	−0.08 to 0.13	0.03	0.07	0.39	0.696	−0.11 to 0.16	0.05	0.08	0.62	0.541	−0.11 to 0.21
	−0.04	0.03	−1.51	0.138	−0.09 to 0.02	0.01	0.03	0.23	0.82	−0.05 to 0.06	−0.02	0.04	−0.57	0.572	−0.09 to 0.05	−0.05	0.04	−1.23	0.224	−0.13 to 0.03

Abbreviation: EDSS, Expanded Disability Status Scale.